POS0409 IMPLICATION OF GLYPICANS AND NOTUM IN BONE MARROW MESENCHYMAL STROMAL CELLS DURING OSTEOGENIC DIFFERENTIATION IN OSTEOARTHRITIC DISEASE
Authors: I. González-Guede, M. López-Ramos, L. Rodriguez Rodriguez, L. Abasolo, B. Fernandez
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Osteoarthritis (OA) is accompanied by an excessive formation of underlying bone caused by homeostatic alterations. It has been described that in OA joints there is an up-regulation of the WNT/β-catenine pathway driving to the differentiation of Mesenchymal Stromal Cells (MSCs) into osteocytes [1]. Glypicans 1-6 act as co-receptors of WNT pathway, improving interactions between molecules [2]. On the other hand, NOTUM plays a role as a negative regulator of the pathway preventing WNT binding to its receptors and favouring the soluble form of Glypicans [3]. All these evidences lead us to think about the importance of Glypicans 1-6 and NOTUM in the osteogenic differentiation of MSCs in OA disease. <h3>Objectives</h3> To determine the differences at gene and protein levels of Glypicans 1-6 and NOTUM in MSCs between OA patients and controls at baseline and during induced <i>in vitro</i> osteogenic differentiation. <h3>Methods</h3> Bone Marrow Mesenchymal Stromal Cells (BM-MSCs) from both, 8 OA patients and 8 healthy donors were isolated from BM samples. BM samples were obtained by joint replacement and traumatic fractures. BM-MSCs were cultured during 21 days with normal culture medium and with osteogenic inducing medium. Cells and supernatant were recovered at 1, 7, 14 and 21 days. Protein levels were determined by ELISA in cells supernatant. Extraction and purification of RNA from cells and cDNA synthesis were performed. Glypicans 1-6 and NOTUM gene-expression was analysed by qPCR and normalized with housekeeping genes β-actin and RNA18S. The data were calculated with the method of fold change (2<sup>-ΔΔCt</sup>). Statical analysis were performed with GraphPad Prism 8.0. For the detection of outliers, the ROUT method (Q=1%) was used. Data were analyzed using t-test. <h3>Results</h3> The mRNA levels of cells with normal culture medium were up-regulated in GPC6 (t=7d; p=0,016) in OA patients. During osteogenic differentiation, was observed a statistically significant up-regulation in levels of OA patients vs controls of GPC2 (t=14d; p=0,034), GPC4 (t=14d, p=0,025; t=21d, p=0,017), GPC5 (t=14d, p=0,007), GPC6 (t=14d, p=0,013) and <i>NOTUM</i> (t=14d, p=0,009) and down-regulation of GPC3 (t=7d, p=0,034). In addition, protein levels of NOTUM were lower compared to the control group in OA BM-MSCs (t=1d, p=0,049) and in the differentiation to osteocytes (t=1d, p=0,049; t=7d, p=0,043; t=14d, p=0,016). GPC3 levels were higher OA patients in all the times and in the two conditions (p &lt; 0,0001). In the differentiation, GPC2 were lower (t=7d, p&lt; 0,0001). GPC1, GPC4 and GPC6 were not present in any case. <h3>Conclusion</h3> Our results evidence a dysregulation in the glypicans in BM-MSCs of OA patients and, specially, during osteogenic differentiation. NOTUM, extracellular negative regulator of the WNT/β-catenine signaling pathway, is decreased at protein level in OA patients, despite the fact that gene expression is elevated. Thus, our data confirm the differences in expression in NOTUM and Glypicans between OA patients and healthy controls. Further studies are needed proposing these molecules, specially NOTUM, as an effective treatment of the disease. <h3>References</h3> [1]Tornero-Esteban <i>et al.</i>, 2015 "Altered Expression" PLoS One. [2]Boudin <i>et al.,</i> 2013 "The role" Semin Arthritis Rheum. [3]Nusse R., 2015 "Cell signalling" Nature. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.